We have purified the unactivated form of the glucocorticoid receptor from rat liver to homogeneity and can prepare 80-100 Hg in a scaled-up purification procedure in one day starting from cytosol. The receptor is homogeneous at 90,000 molecular weight by SDS-gel electrophoresis. Preliminary experiments indicate that the native, homogeneous receptor about 320,000 molecular weight) is activatable to a DNA- or nuclear binding form, but not as extensively as in crude systems. Thiscan be accomplished by heat or high salt and addition of alkaline phosphatase produces an equally activated form after incuibatioon in the cold. Using the homogeneous preparation, we plan to: (1) characterize the factors required for the activation process; (2) determine if the unactivated receptor is phosphorylated and becomes dephosphorylated during activation; (3) determine if pyriodoxal-P interacts directly with the purified receptor and, if so, sequence the pyridoxal-P binding site(s), (4) sequence the major released fragments after cyanogen bromide treatment and partial protiolysis of the receptor and also sequence the steroid and DNA-binding sites, and (5) determine the effects of addition of homogeneous receptor complexes on transcription processes in preparationsfrom anterior pituitary cells capable of transcribing mRNA encoding growth hormone and prolactin. These experiments will add to our understanding of the mechanism of action of the glucocorticoid receptor and its structure and should result in quantities of purified receptor which may be microinjected in targeted liposomes for treatment of glucocorticoid-resistant human leukemias.